The bacterial pathogenesis of Nontypeable H. influenzae (NTHi) otitis media is not well understood. Evidence suggests that bacterial adherence (specific and non-specific interactions) and the delivery of bacterial toxin on the target epithelial or endothelial cells are critical for the pathogenesis of otitis media and virulence factor. The paucity of information makes it difficult to formulate an effective approach to developing a vaccine or reducing the inflammatory response. The objectives are to define: the cellular and molecular mechanisms involved in adherence of NTHi on the respiratory epithelial cells; the role of endotoxin in the pathogenesis of otitis media; the antigenicity to NTHi surface antigens as pathogenic and virulence factors; the role of mucosal immunity in protection and pathogenesis. Quantitation and Biological Properties of Released NTHi LPS: The release of lipooligosaccharide (LOS) from NTHi may play an important role in the pathogenicity of NTHi caused otitis media. The amount of LOS in bacterial cells and the growth medium for five NTHi strains was determined. During a 3-day growth period, the NTHi released variable, but significant amounts of LOS into the growth medium. Biological properties of released and cell-bound LOSs from two representative strains were compared. The released LOS showed approximately a 10-fold increase in inducing human monocytes to produce TNF-a, IL-beta and IL-6; a 16 to 37-fold increase in the clotting of limulus amebocyte lysate (LAL); and a 13 to 28-fold increase in mouse lethal toxicity. These results suggest that the released LOS or its inflammatory mediators may play a more important role than the LOS in the pathogenicity of NTHi caused otitis media. Study of Bacterial Adherence: The goal is to isolate and clone the host receptor for the adhesion molecules expressed on the surface of the bacteria. We are characterizing the rat middle ear epithelial cell line and screening NTHi strains that are highly adherent.